1. Field of the Invention
The invention is directed to transgenic expression of amphipathic and lytic peptides in mammalian organisms, and particularly to transgenic mammalian unicellular and multicellular organisms having stably-integrated amphiphathic peptide-encoding genes whose expression is controlled by a tissue-specific mammalian regulatory sequence.
2. State of the Art
A class of cellular polypeptides known as "lytic peptides" or "amphipathic peptides" has been found to be active against various disease-causing agents including bacteria and viruses. These amphipathic peptides form complexes in the cell's outer coat or membrane, and a present hypothesis is that the complexes form pores which allow unregulated transfer of fluid and molecules across the membrane. According to this hypothesis, the cells die because of osmotic imbalances resulting from this unregulated transfer. Whatever the mechanism, the naturally occurring amphipathic peptides and various modified peptides having certain peptide sequence or structure in common with the naturally occurring amphipathic peptides have been found to have cytocidal activity against bacteria, fungi, protozoans, and various other microbial pathogens.
PCT patent publication No. WO 89/00194 (priority date U.S. patent application filed Jul. 7, 1987), by Jaynes et al., discloses numerous amphipathic peptides including both naturally occurring ones and modified amphipathic peptides. An earlier application by Jaynes et al., U.S. Pat. application Ser. No. 07/889,225 filed Jul. 25, 1986, discloses vectors encoding certain amphipathic peptides and the production of disease-resistant transgenic plants containing gene sequences expressing such amphipathic peptides. The Jaynes applications also disclose use of amphipathic peptides for injection or application to a sick organism.
Quantities of amphipathic peptides or amphipathic peptide analogues sufficient for experimental use can be made on a peptide synthesizer. However, this method is not suitable for commercial production or even for amounts needed for clinical trials of amphipathic peptides as disease-treating agents.
Attempts have been made to produce amphipathic peptides by overproduction in various host species. Amphipathic peptides can be produced in insect cell cultures using the baculovirus-Spodoptera expression system, as the amphipathic peptides are native to these insect species and the insect cells are somewhat resistant to their toxic effects. Efforts to express amphipathic peptides in non-insect hosts have generally worked poorly, because of the toxic effects of the amphipathic peptides on the host cells or organisms. While Jaynes et al. were able to produce transgenic plants expressing an amphipathic peptide, their success is due to the difference in cell wall and membrane structures of plants, which render them insusceptible to attack by the amphipathic peptide. So far as the present inventors are aware, the only successful production of a transgenic mammalian organism containing an expressible lytic peptide gene is that disclosed in the related copending application Ser. No. 08/114,692 of White and Reed. These inventors were able to produce transgenic mice and transformed lymphocytes carrying an integrated lytic peptide-encoding gene under the control of an interleukin regulating sequence. However, these organisms were not particularly suited for achieving large-scale production of amphipathic peptides.
Further, the production of novel organisms by techniques of genetic engineering, including both unicellular and multicellular organisms and tissue culture cell lines, has been applied to achieve various objectives. For example, the "Harvard mouse" is a strain of mice which have been genetically altered to have increased susceptibility to the induction of cancer by damaging to a particular gene. Another example is the development of a bacterial strain carrying a foreign gene which confers the ability to "eat" petroleum and related compounds on the host.
Desirably, in a genetically-engineered organism, the foreign gene should be stably present in the germ cells of the organism so that it is transmitted to its offspring and to subsequent generations of the organism. Further, it is often desirable that the process of obtaining the transgenic organism not require integration of the gene at a specific site in order for expression of the gene to occur. This makes the process more reproducible, which is particularly important when dealing with alteration of multicellular animals where the generation times are generally long compared to those of unicellular organisms.
However, so far as the present inventors are aware, no one has yet been able to achieve a disease-resistant mammalian or other non-insect vertebrate unicellular or multicellular organism, having an expressible gene encoding an amphipathic peptidestably integrated into its genome. While Jaynes et al. were able to produce transgenic plants expressing an amphipathicpeptide, their success may be due to the difference in cell structure of plants, such as the cell wall and membrane structures, which make them insusceptible to attack by the amphipathic peptide. In contrast, in other prokaryotic and non-insect eukaryotic cells and animals, expression of an introduced gene for an amphipathic peptide can result in death or serious harm to the host cell. If the gene encoding the amphipathic peptide is under the control of a regulator that permits even low levels of expression, long-term growth of the host (or culture of the cell line) is difficult to achieve.
While their sensitivity is less than that of bacteria and most eukaryotic pathogens, mammalian cells are also susceptible to killing by amphipathic peptides. Unless the gene encoding the amphipathic peptide is under very tight control, leaky expression of the peptide has generally negative effects on host mammalian cells. However, if tight control is provided, one is faced with the problem of how to obtain selective and beneficial expression of the amphipathic peptide, otherwise the benefits of the integrated gene cannot be realized.
So far as the present inventors are aware, to date there have been no successful attempts to selectively express amphipathic peptides in a beneficial manner in particular mammalian cell types or tissues. Accordingly, a need remains for means to achieve such selective expression of amphipathic peptides in mammalian cells and organisms.
Nor are the inventors aware of any successful attempts to use a non-insect expression system, or a transgenic mammalian peptide expression system, for production of amphipathic peptides in large quantity. Accordingly, a need also remains for means to produce amphipathic peptides in large quantities.
Further, a need remains for a DNA cassette and method for producing mammalian and non-insect eukaryotic transgenic organisms having a stably integrated gene encoding an amphipathic peptide, with the gene being selectively expressed only under conditions such as disease states where expression is desirable and without significantly jeopardizing the general hardiness and well-being of the host organisms. A need further remains for such DNA cassette and method which is useful to transfect both unicellular and multicellular non-insect vertebrate organisms.